Stability indicating potentiometric method for the determination of palonosetron HCl using two different sensors

This paper presents a novel potentiometric approach for the determination of palonosetron HCl using two sensors; ionophore-free and ionophore-doped ones. The two sensors successfully determined the cited drug in the range of 1 × 10–5–1 × 10–2 M with respective Nernstian slopes of 54.9 ± 0.25 and 59.3 ± 0.16 mV/decade. Incorporating calix[8]arene as an ionophore resulted in a lower detection limit (LOD = 3.1 × 10–6 M) and enhanced selectivity when compared to the ionophore-free sensor (LOD = 7.9 × 10–6 M). This modification was also associated with faster response for the ionophore-doped sensor (response time = 20 s) compared to the ionophore-free one (response time = 30 s). The two sensors showed a stable response over a pH range of 3.0–8.0. They successfully determined palonosetron HCl in presence of its oxidative degradation products. They were also used for direct determination of the drug in commercially available parenteral solution without any interference from other dosage forms’ additives.


Results and discussion
Ion-selective membranes are usually prepared from PVC, electroactive substance (ion-association complex) and a plasticizer. The role of PVC is to provide an inert solid support structure in which the rest of components are embedded. The plasticizer dissolves the ion association complex, plasticizes the membrane and affects the lipophilicity of PVC membrane. It also alters the distribution coefficient (K) of different species thus affecting the performance characteristics of electrode 31 . In potentiometric applications, selectivity and sensitivity are the main aim that guide optimization plan during method development. A key component in ISE fabrication that can significantly improve selectivity and sensitivity is doping the PVC polymeric membrane with an ionophore 32 . The response of membranes containing ionophores is largely governed by molecular recognition where the analyte functions as the guest and the ionophore plays the role of host 33 . ISEs containing ionophores have been shown to increase the selectivity of the sensor toward the detection of specific analytes 27 . Calixarenes are widely used as ionophores for various ions via dipole-dipole interactions where they can make stable host-guest inclusion complexes with different types of cation substrates. As a result, they have been largely exploited for the development of a number of ISEs 22,29,34 . Stability study. As per our previously reported work, PALO was subjected to forced acidic, alkaline, oxidative, photolytic and thermal degradation conditions 7 . Observed degradation was noticed only under oxidative condition upon refluxing with 6% H 2 O 2 for 6 h. Three degradation products were separated on HPTLC plate and mass analysis was then conducted by means of Advion compact mass spectrometer 7 . The obtained mass spectra of PALO along with its three oxidative degradation products are shown in Figure S1, supplemental information. As a result and in this work, calix [8]arene was incorporated as an ionophore and the fabricated ISE was compared to the ionophore-free one in terms of sensitivity and selectivity for PALO determination in presence of its oxidative degradation products. The pKa value of PALO is ≈ 8.81, so at pH 5.0, the studied drug has a positive charge. The use of TPB as counter ion for the cationic PALO in the two proposed ion-sensitive membrane sensors was suggested.
Response characteristics of the proposed sensors. The performance characteristics of the two proposed ISEs were assessed according to the IUPAC standards 27 , Table 1. Two calibration curves were constructed, showing the same linearity range; 1 × 10 -5 to 1 × 10 -2 M as displayed in Fig. 1. The obtained slopes were 54.9 ± 0.25 and 59.3 ± 0.16 mV/decade for ionophore-free and ionophore-doped sensors, respectively, with respective detection limits of 7.9 × 10 -6 and 3.1 × 10 -6 M. The enhanced responses of ionophore-doped sensor are attributed to; (1) presence of rigid calix [8]arene cavity which selectively complex with PALO, (2) fast complexation-decomplexation kinetics for reversible transduction processes, and (3) high lipophilicity (especially with 8 phenolic units of calix [8]arene) preventing complexed PALO from leaching from nonpolar membrane into aqueous phase 35 .
Effect of pH. The effect of pH on the response of the proposed sensor is investigated by using 1 × 10 -2 M and 1 × 10 -3 M PALO standard solutions at different pH values, ranging from 3.0-11.0. The obtained potentials were recorded at each pH value. It was found that the investigated electrode showed a stable response over a pH range of 3.0-8.0, Fig. 2.

Selectivity of the proposed sensors. Selectivity of proposed PALO sensors in presence of interfering
ions was evaluated by separate solutions method. Four inorganic ions; Na + (as citrate), K + , Ca 2+ and Mg 2+ (as chlorides), oxidative degradation products as well as structurally related granisetron were selected for this study. The two proposed sensors showed non-Nernstian responses to the inorganic ions, Fig. 4. This is attributed to the hydrophobic nature of ion selective membrane which hinders the hydrophilic inorganic ions exchange. As a result, no need to determine selectivity coefficients for those ions 36 . On the other hand, oxidative degradation products and structurally related drugs (granisetron & ondansetron) showed near-Nernstian responses to monovalent cations (≈ 45 mV/decade) over the considered range, Fig. 4. Selectivity coefficients were consequently calculated, Table 2. As shown, about one order of magnitude enhancement was noticed for ionophore-doped sensor. This supports its exceedances over ionophore-free one as stability-indicating sensor. In nutshell, incorporation of calix [8]arene, during fabrication of ISE, improved the selectivity of the proposed ionophore-doped sensor as compared to the ionophore-free one.
Application to dosage form. The two proposed sensors were successfully applied for the determination of PALO in Emegrand vial, Table 1. It is worth noting that this potentiometric method offers the advantage of direct PALO determination without any pretreatment steps.  The calculated values of t and F are less than their corresponding tabulated ones, which reveals that there is no significant difference between the suggested and official method with respect to accuracy and precision, Table 3. Moreover, a point-by-point comparison with the two reported stability-indicating chromatographic methods was conducted, Table 4. Besides the no need for preliminary drug preparation, our proposed sensors achieved a comparable quantification limit (LOQ) as well as ability of PALO determination in presence of other structurally related drugs.

Conclusions
This work demonstrates the first potentiometric method for palonosetron HCl determination. Two ion-selective electrodes were fabricated; one without incorporation of ionophore (ionophore-free senor) and the second one utilizing calix [8]arene as an ionophore (ionophore-doped senor). The incorporation of calix [8]arene, as an ionophore, improved the limit of detection as well as the selectivity of the proposed sensor towards the most likely formed degradation products and the structurally related drugs. Selectivity assessment revealed that the fabricated sensors could be applied as stability-indicating ones and in direct determination of the cited drug in presence of its dosage form additives. The proposed method was found to be sensitive, rapid, easy to use, selective, simple and more economic for palonosetron HCl determination as compared to other reported ones. The method showed also good applicability for determination of the cited drug in its marketed dosage form (Emegrand vials) promoting its use in different quality control laboratories. Instrumentation. Ag/AgCl reference electrode (Thermo Scientific Orion 90-02, MA, USA). pH meter for pH adjustments and potential measurements (Hanna 211). Magnetic stirrer (Bandelin Sonorex, Rx 5105). HPTLC aluminum plates 10 × 10 cm precoated with 0.25 mm silica gel 60 F 254 (Merck, Germany) were used for stability study. The plates were developed at ambient temperature using a mixture of methanol: ammonia (10: 0.5, v/v) as the developing system in a CAMAG twin-trough chamber previously saturated with the developing system for 30 min. Advion compact mass spectrometer (CMS, USA) provided with ESI ion source was utilized for mass analysis.    Sensors calibration. Calibration of the conditioned PALO sensors is performed by immersing them separately in conjunction with double junction Ag/AgCl reference electrode in different PALO standard solutions (1 × 10 -5 to 1 × 10 -2 M) prepared in buffer pH 5.0. The ISEs were allowed to equilibrate while stirring and potential difference (emf) readings were the recorded (within ± 1 mV). The membrane sensors were washed between measurements with the buffer. The recorded potentials were finally plotted as a function of logarithm PALO concentrations in buffer pH 5.0 at 25 °C. A diagram for measurement process is shown in Fig. 5.

Materials and reagents.
Selectivity studies. The potentiometric selectivity coefficients ( K pot PALO,Int ) of the proposed ISEs were evaluated according to IUPAC 27 guidelines using the separate solutions method 39,40 . Calibration curves for some inorganic cations, oxidative degradation products as well as a structurally related organic ions, granisetron and ondansetron, were constructed using the two proposed sensors. Potentials for the same concentration (1 × 10 -4 M) of PALO cation and interfering ions were measured separately, and the rearranged Nicolsky-Eisenman equation was applied 36,39 .
where E A and E B are potentials measured for ion of interest (with Z A charge) and interfering ion (with Z B charge), respectively, and (2.303RT/Z A F) is the slope of the calibration curve in mV/decade. Application to pharmaceutical dosage form. Five vials of Emegrand were emptied and transferred into a 50-ml volumetric flask. The volume was completed to the mark with Robinson buffer pH 5.0 to prepare a solution of 8.4 × 10 -4 M PALO. The emfs produced by immersing the prepared electrode in conjunction with the double junction Ag/AgCl reference electrode in the prepared solution at 25 °C were recorded. The concentration of PALO was calculated from the following regression equations: where Y is the potential in mV and X is the logarithm of the concentration in M.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.